Viroids and Rootstocks on Field Performance of Tahiti Acid Lime

Article/Artigo Citrus Res. Technol., 41, e1063, 2020 ISSN 2236-3122 (Online) https://doi.org/10.4322/crt.20420

Viroids and rootstocks on field performance of Tahiti acid lime

Eduardo Sanches Stuchi1,2*, Simone Rodrigues da Silva3, Otávio Ricardo Sempionato2, Eduardo Toller Reiff2, Luiz Gustavo Parolin2 & Dilermando Perecin4

SUMMARY

Tahiti acid lime [Citrus latifolia (Yu.Tanaka) Tanaka] is relevant in the Brazilian citrus industry, and it represents 7.5% of the total world production of lemons and acid limes. Here, the effects of two citrus viroids inoculation and six rootstocks on the vegetative growth and yield of Tahiti acid lime [Citrus latifolia (Yu.Tanaka) Tanaka] were evaluated: (i) FCAV and (ii) Rubidoux trifoliate orange [Poncirus trifoliata (L.) Raf.], (iii) FCAV and (iv) Limeira Rangpur lime [Citrus limonia (L.) Osbeck], (v) Carrizo citrange [C. sinensis (L.) Obsbeck x Poncirus trifoliata (L.) Raf.] and (vi) Sunki mandarin [Citrus sunki Hort. ex Tanaka]) were inoculated with the viroids mixtures [CEVd + HSVd (a non-cachexia variant) + CDVd] and [HSVd (a non-cachexia variant) + CDVd]. Non-inoculated trees of each rootstock were used as control. The experimental design was randomized blocks in a split-plot scheme with three replications and one plant per plot. The inoculation was done under field conditions using infected bud and grafting ten months after planting. From the 33 months after inoculation, both viroid mixtures reduced tree growth of all rootstocks except Sunki. Trees grafted on trifoliate oranges rootstocks were less vigorous 55 months after inoculation. From 36 to 60 months after planting, the yield of non-inoculated trees was significantly higher than inoculated ones, regardless of the rootstock. Scion trees grafted on FCAV and Limeira Rangpur lime showed higher yields. Viroids mixtures did not affect drought tolerance. Scion trees grafted on the Rangpur lime clones or Sunki mandarin showed the highest drought tolerance. In conclusion, there was no evidence of yield and drought tolerance advantage, neither production of out-of-season fruits compared to the non inoculated control in the use of the viroids mixtures evaluated for Tahiti lime plantings. Index terms: Citrus spp, fruit yield, exocortis, tree size control, planting density.

Desempenho de limeira ácida Tahiti em função de viroides e porta-enxertos

RESUMO

A lima ácida Tahiti [Citrus latifolia (Yu.Tanaka) Tanaka] apresenta grande importância na citricultura brasileira, representando 7,5% da produção total de limas ácidas e limões. Neste estudo, foram avaliados os efeitos da inoculação de viróides dos citros e de porta-enxertos no crescimento

1 Embrapa Mandioca e Fruticultura, Cruz das Almas, BA, Brasil 2 Estação Experimental do Agronegócio de Bebedouro, Bebedouro, SP, Brasil 3 Universidade de São Paulo – USP, Campus de Piracicaba, Piracicaba, SP, Brasil 4 USP/ESALQ, Piracicaba, SP, Brasil Autor de correspondencia: Eduardo Sanches Stuchi, Estação Experimental do Agronegócio de Bebedouro, Rodovia Faria Lima, Km 384, CEP 14713-000, Bebedouro, SP, Brasil. E-mail: [email protected]

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Stuchi et al. 2/12 vegetativo, produtividade da lima ácida Tahiti [Citrus latifolia (Yu.Tanaka) Tanaka]. Plantas da cultivar enxertadas em seis cultivares de porta-enxertos: trifoliatas FCAV e Rubidoux [Poncirus trifoliata (L.) Raf.], limoeiro Cravo FCAV e Limeira [Citrus limonia (L.) Osbeck], citrangeiro Carrizo [C. sinensis (L.) Obsbeck x Poncirus trifoliata (L.) Raf.] e tangerineira Sunki [Citrus sunki Hort. ex Tanaka]) foram inoculadas com duas misturas diferentes de viróides compostos de combinações de espécies e viróides: 1) CEVd) + HSVd (variante não-cachexia) + CDVd; e 2) HSVd (variante não cachexia) + CDVd. Árvores não-noculadas de cada porta-enxerto foram utilizadas como controle. As inoculações foram realizadas em condições de campo por enxertia de borbulhas, dez meses após o plantio. A partir do 33º mês após a inoculação, os dois isolados de viróides reduziram o crescimento das árvores em todos os porta-enxertos, exceto a tangerineira Sunki. As plantas enxertadas nos porta-enxertos de trifoliata foram menos vigorosas 55 meses após a inoculação. De 2004 a 2006 (36 a 60 meses após o plantio), a produção de frutos das plantas não inoculadas foi significativamente maior do que a das inoculadas. A limeira ácida Tahiti enxertada em FCAV e Limeira Cravos apresentou altos rendimentos em frutos. As misturas de isolados de viróides não tiveram efeito na tolerância à seca. Plantas sobre porta-enxerto de limoeiro Cravo ou em tangerineira Sunki apresentaram a maior tolerância à seca. Concluiu-se que não houve evidência de vantagem na produção de frutos em época normal e extemporânea e tolerância à seca no uso das misturas de viróides estudadas para plantios de lima ácida Tahiti. Palavras-chave: Citrus spp, produção de frutos, exocorte, controle do tamanho de plantas, densidade de plantio.

INTRODUCTION longitudinal cracks or sunken areas in the bark of the trunk and branches in five of six studied clones. The exocortis- The Tahiti acid lime [Citrus latifolia (Yu.Tanaka) free clone was then named IA-5 (Bearss lime). Tanaka] is cultivated in around 48,000 ha in Brazil. In Sao The same clones were tested by Figueiredo et al. Paulo state there is a production area of about 26,000 ha (1976) and the absence of exocortis was confirmed in (IBGE, 2019). The Brazilian annual production, with the IAC 5, the most productive one. The second position approximately 1.3 million tons, is the fourth largest in the in yield was recorded for the IAC 2 plants, which had world, which represents 7.5% of the total production of maximum production for exocortis-infected clones. lemon/acid limes (FAO, 2019). Its fresh fruit exportation The clones IAC 1, IAC 3, and IAC 4 behaved in an reached US$ 89 million in 2016 (FAO, 2019). intermediate way. The distribution of fruit production The two major clones of Tahiti acid lime cultivated in along the year of IAC 1 was the most favorable and Brazil are IAC-5 (a line free of viroids and also known as IAC 2 had the worst. Peruano) and Quebra-Galho, having almost all plants the Probably, Quebra-galho clone budwood is derivated Rangpur lime [Citrus limonia (L.) Osbeck] as rootstock from the IAC clones aforementioned, except IAC 5, without (Figueiredo; Stuchi; 2003). mother plants or breeding programs reported. Thus, the Quebra-Galho clone is naturally infected by the Citrus Quebra-galho multiplication can be considered clandestine exocortis viroid - CEVd (commonly related to the exocortis and could affect another citrus more sensitive to exocortis disease), alone or in mixture with other viroids as Hop as trifoliate orange and Rangpur lime rootstocks. On the stunt viroid - HSVd and Citrus dwarfing viroid - CDVd other hand, no cachexia/xyloporosis, psorosis, or severe (Eiras et al., 2010). The infected trees show differences tristeza was found in Quebra-galho plants from differents in size and shape of the canopy, and yield, compared to farms in northern São Paulo state (Silva, 2007). healthy IAC-5 trees (Targon et al., 2006). Infected trees Also, the viroids (i.e. Citrus exocortis viroid) are of Quebra-Galho acid lime clone have early production reported as drought-tolerance inducers (Moreira, 1956, and usually give some out-of-season fruits (Figueiredo; Salibe, 1986; Müller and Costa, 1993; Rodriguez et al., Stuchi; 2003). The name Quebra-Galho (Break-Branch) 1974; Pompeu Júnior et al., 1976), earlier and out-of-season derives from the bark-cracking symptom which results in fruit production inducers and tree-size control agents. brittle branches, probably associated with viroid infection These characteristics could be explored in high plantings (Eiras et al., 2010). densities (Bar-Joseph, 1993; Broadbent et al., 1994; Van The presence of exocortis in Tahiti acid lime in Vuuren & Graça, 1997a, b; Semancik et al., 1997, 2002; Brazil was firstly reported by Salibe & Moreira (1965). Stuchi et al., 2007; Vernière et al., 2006; Vidalakis et al., The viroid presence was associated with the development of 2010, 2011; Murcia et al., 2015), which may provide a

Citrus Res. Technol., 41, e1063, 2020 Viroids and rootstocks on field performance... 3/12 higher yield (t ha-1) than conventional plantings densities trifoliata (L.) Raf.], and Sunki mandarin [Citrus sunki (Tucker et al., 1991; Wheaton et al., 1991). In Australia, Hort. ex Tanaka]} were evaluated. CVd-IIIb inoculation is a recommended horticultural practice for dwarfing in high density plantings and is based on officially published guidelines (Hardy et al., 2007). Viroids and inoculation description To achieve a better comprehension of viroids infecting Tahiti acid lime, we assessed the effects of two well-known Trees were inoculated with two viroids mixtures, citrus viroids mixtures, composed of viroids species and composed of viroids species and variant combinations: variant combinations, on the vegetative growth (tree size), 1) Citrus exocortis viroid (CEVd) + Hop stunt viroid yield, out-of-season fruit production, and drought tolerance (HSVd - CVd-II, a non-cachexia variant) + Citrus of Tahiti acid lime trees grafted onto six rootstocks. dwarfing viroid (CDVd), and 2) Hop stunt viroid (HSVd - CVd-II, a non-cachexia variant) + Citrus dwarfing viroid. The inoculation was carried out as described by Stuchi et al. (2007): trees were inoculated in field MATERIAL AND METHODS conditions ten months after planting by grafting of two infected buds per tree one in the scion trunk and another Experimental site in the rootstock trunk. After sprouting, buds were blinded. Non-inoculated plants of each rootstock were used as The experiment was installed in February 2001 in control. The two evaluated mixtures containing CEVd Bebedouro (Lat. 20º 53’ 16” S; Long. 48º 28’ 11” W, 601 m (1) and without CEVd (2) were previously reported as above sea level), Sao Paulo State, Brazil. The prevailing severe and mild, respectively (Pompeu Júnior et al., 1976). climate in the region is Aw (Köppen), with mild and dry The mixtures were characterized according to biological winter and hot and rainy summer, annual mean rainfall of and biochemical indexing, and the genome viroids were 1,420 mm. The averages temperatures for the evaluation previously sequenced (Stuchi et al., 1998; Targon et al., period were 17.3 ºC (minimum), 23.7 ºC (mean) and 2001) and registered in the GenBank under code numbers 30.2 ºC (maximum). The soil was a typical dystrophic red AF434678 (CEVd), AF434679 (HSVd, a non-cachexia latosol, endoallic, acid soil, with a moderate A-horizon and variant), and AF434680 (CVd-III). medium texture (38% clay content; pH CaCl2 = 5.6 and -3 CEC = 51 mmolc dm at 0-20 cm soil depth). Standard cultural practices for Tahiti lime cultivation in São Tree size and yield Paulo state were adopted and the trees were not pruned. The trees were arranged in a spacing of 7 m (between Tree height and canopy diameter were evaluated in rows) per 3 m (between trees) (476 trees ha-1) with no 2002, 2004, 2005, and 2006. Canopy volume was estimated supplemental irrigation. based on the following equation: V= 2/3.Π.R2.H, where V corresponds to canopy volume (m3), R, radius (m), and H, tree height (m) (Pompeu Júnior et al., 1976). The total Plant material and experimental design weight of fruit per tree was measured for all plants between 2003 and 2006. The crop efficiency (kg m-3) The experiment was designed in randomized blocks was estimated by dividing the tree yield (kg tree-1) by the in a split-plot form with three replications and six main canopy volume (m3) from 2004 to 2006. The annual yield treatments (rootstocks), three secondary ones (inoculation from 2003 to 2006 was split into two groups, according and non-inoculation of viroids) with three plants per plot to the production season throughout the year (first and (rootstock), and one plant per subplot (viroid inoculation). second semester), and the data analysis was performed Healthy nursery trees of Tahiti acid lime IAC 5-1 (Bremer according to split-split plot in time design. The theoretical Neto et al., 2013) [Citrus latifolia (Yu.Tanaka) Tanaka]) optimal planting density and estimated potential yields grafted onto six rootstocks {FCAV and Rubidoux were based on the assumption that no reduction in size trifoliate oranges [Poncirus trifoliata (L.) Raf.], FCAV or yield would occur due to closer spacings (Pompeu and Limeira Rangpur limes [Citrus limonia (L.) Osbeck], Júnior et al., 1976). The theoretical optimal spacing for the Carrizocitrange [C. sinensis (L.) Obsbeck x Poncirus trees was calculated on the basis that adjacent trees should

Citrus Res. Technol., 41, e1063, 2020 Stuchi et al. 4/12 overlap 15% in the row and that should be 2.5 meters different mixtures during the evaluation period, and after of open space between the rows (De Negri et al., 2005). 2003 inoculated trees showed lower yield than control ones (Table 2). The canopy growth was restrained when the plants Drought tolerance were inoculated with both mixtures and mainly with CEVd presence. Conversely, no effect was observed in drought The plant’s drought tolerance was determined by tolerance, regardless of the viroid inoculation. Comparing visual evaluation of leaf wilting intensity after a long the rootstocks, Tahiti acid lime on Limeira Rangpur lime drought period in September 2007 using a descriptive plants showed the highest drought tolerance, followed by scale composed by three levels: 1- poor drought tolerance that on FCAV Rangpur lime and Sunki mandarin. Carrizo (leaves with a severe wilting degree and moderate to severe citrange and the two clones of its parental trifoliata orange leaf loss), 2- intermediate drought tolerance (intermediate presented the lowest drought tolerance. None interaction leaf wilting degree and light to medium leaf loss), 3 – between rootstocks and mixtures occurred for drought good tolerance (low leaf wilting degree and without or tolerance assessment (Table 2). with light leaf loss) (Stuchi et al. 2000). The effect of inoculation with viroids on the accumulated Results were submitted to analysis of variance and production of Tahiti acid lime plants varied according to the means were compared by the Tukey test (P<0.05). the rootstock variety. FCAV and Limeira Rangpur lime For drought tolerance data, the non-parametric analysis stood out as good yielders followed by FCAV trifoliate using the Kruskal-Wallis test was performed and means orange (Table 3). While for all other rootstocks, both viroids were compared by Dunn’s multiple comparison test mixtures decreased the production, for Sunki mandarin (P<0.05). Statistical analyzes were performed using the there were no differences between these treatments and SAS statistical package (SAS Institute Inc, 1995). non-inoculated ones. Sunki mandarin and Tahiti acid lime were reported as asymptomatic for viroids infection after inoculation, showed S-PAGE negative results, and very RESULTS AND DISCUSSION low titer of CEVd by molecular hybridization analysis of bark samples (Barbosa et al., 2002). The two species’ The effect of both viroids mixtures on canopy reactions to CVd-II and CVd-III by S-PAGE were also diameter and tree height was observed from 33 months positive. However, Sunki mandarin expresses low titer for after inoculation. In the last evaluation, 55 months after both viroids, while Tahiti acid lime presented very high inoculation, the average reduction was 17% and 10% for CVd-III titer besides symptoms (Barbosa et al., 2002). canopy diameter and 26% and 16% for plant height of In our results, tree size-reduction appears to be due to plants inoculated with and without CEVd, respectively, in rootstock cultivars susceptibility more than to the scion relation to control plants. Reductions of 48% and 33% in cultivar considering that the inoculated trees had very canopy volume for the mixtures with and without CEVd, similar plant height and canopy diameter to control ones. respectively, related to non-inoculated plants were also We observed symptoms of bark cracking on Tahiti acid observed in the same evaluation, with the mixture with lime branches grafted onto Sunki mandarin inoculated CEVd differing from the without CEVD one. Plants only with the viroid CEVd + HSVd + CVd-III mixture, grafted on Carrizo citrange, FCAV Rangpur lime, and but Sunki mandarin rootstock presented no symptoms in Limeira Rangpur lime rootstocks induced higher canopy the trunk 55 months after inoculation. On the other hand, volume than plants grafted on both trifoliate orange Pompeu Junior (2005) commented about Sunki mandarin cultivars (Table 1). intolerance to exocortis. Limeira and FCAV Rangpur lime rootstocks induced Tahiti acid lime trees showed differences in crop the highest fruit production in the first crop year (2003, efficiency due to rootstocks only in 2005 when FCAV 24 months after planting, 36 months after inoculation) but trifoliate orange outstood comparing to Carrizo citrange. did not differ from Rubidoux trifoliate orange. Although In 2006, trees inoculated with the CEVd mixture presented statistically similar, the yield of Tahiti trees grafted on crop efficiency higher than that ones infected without Limeira and FCAV Rangpur limes, were respectively the CEVd mixture but similar to non-inoculated trees 26 and 28% higher than that of FCAV trifoliate orange. (Table 4). The efficiency for all treatments was inferior No differences occurred between plants inoculated with to those reported for Tahiti IAC 5 grafted on common

Citrus Res. Technol., 41, e1063, 2020 Viroids and rootstocks on field performance... 5/12 a c a bc ab a c b a ) 3 (m Volume Volume 31.62 17.95 30.70 18.69 27.66 30.73 18.64 24.13 35.91 a b a b a b c b a 55 (m) Height 3.31 2.73 3.37 2.73 3.31 2.95 2.65 2.99 3.56 a c a bc ab a c b a (m) 4.18 3.37 4.14 3.46 3.97 4.19 3.53 3.85 4.27 Diameter ≤ 0.05) p a c ab bc abc ab b b a ) 3 (m Volume Volume 25.61 15.91 23.51 17.73 21.36 23.57 16.77 20.20 26.88 A B A ab ab ab C B A 41 (m) Height 3.16 2.61 3.17 2.73 3.06 2.98 2.59 2.90 3.36 a b ab ab ab a b ab a (m) 3.82 3.30 3.74 3.42 3.64 3.81 3.40 3.59 3.88 Diameter a c ab bc ab a b b a ) 3 (m Volume Volume 11.08 16.76 10.32 14.73 15.13 15.68 10.89 13.34 17.63 a c ab bc ab ab c b a 33 (m) Height 2.97 2.48 2.85 2.59 2.87 2.83 2.45 2.71 3.13 ) of ‘Tahiti’ acid lime on six rootstocks inoculated or not with viroids at different ages. acid lime on six rootstocks inoculated or not with viroids at different ) of ‘Tahiti’ 3 a b ab b a a b b a (m) 3.21 2.76 3.12 2.79 3.17 3.20 2.84 3.02 3.26 Diameter a a a a a a a a a (m) 1.86 1.74 1.83 1.80 1.92 1.81 1.82 1.81 1.84 Height 1 12 a a a a a a a a a (m) 2.01 1.96 1.93 1.77 1.89 1.88 1.87 1.86 2.00 Diameter Treatments Canopy diameter (m), height and volume (m Months after inoculation Means followed by the same lower letter case in the column comparing rootstocks or mixtures do not differ by the Tukey test ( Tukey by the Means followed by the same lower letter case in column comparing rootstocks or mixtures do not differ Rootstocks Carrizo citrange Rubidoux trifoliate orange Limeira Rangpur lime trifoliate orange FCAV Sunki mandarin Rangpur lime FCAV Mixtures Viroid CEVd + CVd-II CV-III CVd-II + CV-III Control Table 1. Table Bebedouro, Brazil 1

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Table 2. Annual yield (kg tree-1) and drought tolerance of ‘Tahiti’ acid lime grafted on six rootstocks from 2003 to 2006, inoculated (2001) or not with different viroids mixtures. Bebedouro, Brazil Years Drought Treatments 2003 2004 2005 2006 Tolerance1 Rootstocks Carrizo citrange 3.91 c2 17.22 b 47.02 b 40.12 abc 1.33 bc Rubidoux Trifoliate orange 8.59 abc 23.83 ab 47.07 b 29.16 c 1.11 c Limeira Rangpur lime 13.24 a 33.88 a 63.23 ab 49.51 a 2.67 a FCAV trifoliate orange 7.37 c 25.47 ab 60.19 ab 33.84 bc 1.11 c Sunki mandarin 7.89 bc 29.08 ab 47.29 ab 33.40 bc 2.22 ab FCAV Rangpur lime 12.62 ab 36.64 a 67.41 a 45.88 ab 2.22 ab Viroid Mixtures CEVd + CVd-II + CVd-III 11.02 a 23.71 b 37.36 b 31.19 b 1.67 a CVd-II + CVd-III 7.27 a 21.09 b 46.63 b 28.53 b 1.72 a Non-inoculated 8.52 a 38.26 a 82.12 a 56.23 a 1.94 a 1 Based on a descriptive scale of drought tolerance: 1- poor tolerance, 2- intermediate tolerance, 3 – good tolerance in 2007; 2 Means followed by the same lower case letter in the column do not differ by the Tukey test (p ≤ 0.05).

Table 3. Interaction effect between six rootstocks and viroids mixtures on the cumulative yield (kg tree-1) of ‘Tahiti’ acid lime from 2003 to 2006. Bebedouro, Brazil Viroid Mixtures CEVd + CVd-II + Rootocks Averages Rootstocks CVd-II + CVd-III Non-inoculated CVd-III kg tree-1 Carrizo citrange 58.63 bB1 89.17 aB 177.03 abA 108.28 b Rubidoux trifoliate orange 58.45 bB 84.99 aB 182.51 abA 108.65 b Limeira Rangpur lime 121.13 abB 131.18 aB 227.29aA 159.87 a FCAV trifoliate orange 107.80 abB 95.76 aB 177.05 abA 126.87 ab Sunki mandarin 121.28 abA 108.67 aA 123.00bA 117.65 b FCAV Rangpur lime 152.42 aB 111.41 aB 223.83aA 162.55 a Viroids Mixtures Averages 103.29 B 103.53 B 185.12A 1 Means followed by the same lower case letter in the column and same capital in the line do not differ by the Tukey test (p ≤ 0.05) Table 4. Crop efficiency of ‘Tahiti’ acid lime on six rootstocks inoculated with viroids. Period of evaluation: 2004- 2006. Bebedouro, Brazil Year 2004 2005 2006 Treatments kg m3 Rootstocks Carrizo citrange 1.05 a1 1.85 b 1.16 a Rubidoux trifoliate orange 2.18 a 2.72 ab 1.77 a Limeira Rangpur lime 2.3 8 a 2.80 ab 1.68 a FCAV trifoliate orange 2.52 a 3.28 a 2.28 a Sunki mandarin 1.91 a 2.24 ab 1.32 a FCAV Rangpur lime 2.47 a 2.98 ab 1.55 a Viroid Mixtures CEVd + CVd-II + CVd-III 2.34 a 2.41 a 1.99 a CVd-II + CVd-III 1.70 a 2.40 a 1.28 b Control 2.23 a 3.13 a 1.61 ab 1 Means followed by the same lower case letter in the column do not differ by the Tukey test (p ≤ 0.05).

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Rangpur lime and Davis A trifoliate orange, 4.1 kg The effects of the viroids HSVd and CDVd on the m3 (Espinoza-Núñez et al., 2011), but quite similar to IAC canopy diameter, tree height, and canopy volume were 5-1 budded onto Swingle citrumelo, 1.50 kg m3 (Bremer observed from 33 months after inoculation, 15 months Neto et al., 2013). earlier than observed by Broadbent et al. (1994) in an The production of out-of-season fruits (in the second experiment carried out in Australia with those viroids semester in Brazil) was similar for all rootstocks, nearly in which reduction in Valencia sweet orange growth 52% of the total yield. The CEVd mixture inoculated trees was detected only at 48 months after inoculation. showed similar fruit production in the two semesters of The differences between inoculated and non-inoculated the year, but those inoculated with the mixture without plants were observed 55 months after inoculation. It has CEVd had the greatest yield in the first semester, fact occurred 23 months earlier than in the inoculation of commercially not interesting. Finally, control plants Marsh Seedless grapefruit (Citrus paradisi MacFayden) produced more fruit in the second semester than the on trifoliate orange (Stuchi et al., 2007). Those differences inoculated ones (Table 5). Figueiredo et al. (1976) related could be attributed to climate conditions and species and that not all exocortis infected clones had good yields or cultivars’ genetic responses to viroids. more favorable distribution of production throughout the The inoculation with the CEVd mixture provoked a year (more production of out-of-season fruits). dwarfing effect corresponding to a 48% reduction in canopy In our study, we evaluated the effects of two viroids volume and 0.9 m in height compared to non-inoculated mixtures on the vegetative growth and fruit production of plants for all rootstocks in average. This reduction differed Tahiti lime grafted on six different rootstocks, five of which from studies carried out in Australia with the cultivars being considered susceptible to Citrus exocortis viroid Bellamy and Washington navel oranges, Valencia sweet (Roistacher, 1991). Rangpur lime and Carrizo citrange orange, Marsh Seedless grapefruit (Broadbent et al., 1994; induce more vigorous plant growth than on Rubidoux and Forsyth et al., 1993) and in Brazil for Marsh Seedless FCAV trifoliate orange rootstocks. The plant size results grapefruit (Stuchi et al., 2007), wherein a similar mixture were in agreement with those reported by Figueiredo et al. caused canopy volume reduction of 68 to 84% related (2000, 2001) and Stenzel & Neves (2004). Trifoliate orange to non-inoculated control. On the other hand, inoculated cultivars can be alternative rootstocks to Rangpur lime plants with HSVd and CDVd mixture showed fewer for Tahiti lime as showed previously by Figueiredo et al. size reductions (33%) than observed in Australia (50%) (2000, 2001, 2002). Sunki mandarin showed tolerance (Gillings et al., 1991). Vidalakis et al. (2010, 2011) reported to the viroids mixtures which are not carriers of HSVd the little effect of CVd-IIa and CVd-IIIb mixture on the cachexia variants since the tree size was not affected and performance of Clementine mandarin grafted onto Carrizo no trunk symptoms were observed. citrange and 50% of canopy reduction of Washington navel

Table 5. Fruit production of ‘Tahiti’ acid lime on six rootstocks inoculated with viroids distributed by semesters of the year. Period of evaluation: 2004-2006. Bebedouro, Brazil I semester II semester I/II yield Treatments (January to June) (July to December) kg tree-1% Rootstocks Carrizo citrange 92,02 aA1 84,49 abcA 47,9 Rubidoux trifoliate orange 106,40 aA 93,62 abA 46,8 Limeira Rangpur lime 89,90 bA 124,06 aA 58,0 FCAV trifoliate orange 31,88 bA 39,03 dA 55,0 Sunki mandarin 47,53 bA 45,23 cdA 48,8 FCAV Rangpur lime 41,84 bA 51,61 bcdA 55,2 Viroid Mixtures CEVd + CVd-II + CVd-III 60,58 bA 64,04 bA 51,4 CVd-II + CVd-III 89,15 aA 63,58 bB 41,6 Control 55,05 bB 93,40 aA 62,9 1 Means followed by the same lower case letter in the column and same capital in the line do not differ by the Tukey test (p ≤ 0.05).

Citrus Res. Technol., 41, e1063, 2020 Stuchi et al. 8/12 grafted onto trifoliate orange cv. Rich 16-6 inoculated CEVd + CVd-II + CVd-III but no differences in scion with CDVd in comparison to viroid-free plants, probably plants grafted on Carrizo citrange. due to rootstocks reaction to viroids as observed here for Results obtained in the present study for fruit production citange Carrizo and trifoliate orange clones. efficiency agree with results reported by Forsyth et al. Our results showed a similar size reduction range, (1993) and Broadbent et al. (1994) who showed similar from 10 to 50%, as reported by Salibe (1986) for Tahiti crop efficiency for inoculated plants versus non-inoculated lime plants infected by mild mixtures of exocortis and ones in their reviews on several experiments involving also similar to those reported by Pérez et al. (2005) with viroids inoculation, carried out in Australia with the sweet Persian limes on Citrus macrophylla inoculated with a oranges Bellamy and Washington navels, Valencia and mixture containing CEVd, HSVd, and CVd III – nearby Marsh grapefruit and, by Aranguren et al. (2004) with 44% of size reduction. Yield reduction was around 44% Tahiti limes grafted onto Citrus macrophylla plants for plants with CVd-II and CVd-III mixture and for those inoculated with four viroids mixtures. On the other hand, with CEVd, HSVd, and CDVd-III mixture. This reduction crop efficiency was superior to the control in a series of was proportional to the reduction in canopy volume as studies, which used the same viroid mixtures (Van Vuuren previously reported (Broadbent et al., 1994). & Graça, 1997a, b; Semancik et al. 1997, Pompeu Júnior Plants of Clementine grafted onto trifoliate orange et al., 1976). The rootstock effect on crop efficiency was infected by CEVd + CVd-II + CVd-III showed a higher lower than those reported by Stenzel & Neves (2004) and reduction in 10 seasons for cumulative yield (53%) than Figueiredo et al. (2000, 2001). those infected with CVd-II + CVd-III (31%) (Vernière et al., Our results show that the drought tolerance of 2006). Those reductions are similar to our results, which inoculated and non-inoculated plants was similar, were higher (63%) than results reported by Davino et al. indicating that viroids inoculation did not influence the (2005) for Navelina ISA 315 sweet orange inoculated with genotype drought tolerance as suggested in other studies a mixture carrying CEVd + CVd-II + CVd-III. On the (Rodriguez & Inforzato, 1968; Rodriguez et al., 1974; other hand, viroid inoculation did not induce a 30% to Pompeu Júnior et al., 1976). Inoculated plants showed crop 40% increase in the yield of Tahiti lime as reported by efficiency and fruit quality similar to the non-inoculated Salibe (1986). plants. Furthermore, the viroid interference in the tree Stuchi et al. (2007) reported that the average fruit size leads to lower yields per tree. yield of Marsh Seedless grapefruit grafted onto trifoliate In this way, the use of these viroids is of limited orange relative to non-inoculated plants was 66% lower value because the observed reduction in canopy for mixture without CEVd and 77% lower for the mixture diameter allows increasing planting density to 553 and with CEVd, the same viroid mixtures used in this study. 481 trees ha-1 for trees, calculated as proposed by De Aranguren et al. (2004) found that Tahiti limes grafted Negri et al.(2005), for inoculated mixtures with and onto Citrus macrophylla inoculated with two viroids without CEVd, respectively, resulting in a projected mixtures carrying CEVd + CVd-IIa and CEVd + CVd-III production of 57.1 and 49.8 t ha-1, values inferior showed yield values similar to those obtained from non- to the 88,1 t ha-1 projected to the non inoculated inoculated plants, differently from our results. treatments at the actual tree spacing evaluated [7 m However, the reduction in fruit yield obtained in the per 3 m = 476 trees ha-1). The climate conditions as present research was similar to the lowest reduction in fruit high temperatures and lack of irrigation may help yield (40%) reported by Pérez et al. (2005) in a study with explain once that most viroids studies have been Persian limes grafted onto Citrus macrophylla inoculated carried out in irrigated groves. The beneficial effect of with a mixture carrying CEVd + HSVd + CVd-III. irrigation to increase fruit yield of different clones of Figueiredo et al. (2000, 2001) reported intermediate Tahiti acid lime trees on citrumelo Swingle rootstock to good yield for Rangpur lime and trifoliate orange as was described (Bremer Neto et al., 2013) and viroids rootstocks for Tahiti acid limes, respectively. Rangpur replicate, accumulate and induce in the host, their lime has also been reported as inductive good performance symptoms much more efficiently at elevated temperatures in the study carried out by Stenzel & Neves (2004). (30 to 33 °C) and high intensities of light like occurs Davino et al. (2005) also found differences in the in the experimental site (Duran-Vila et al., 1988). cumulative yield of Navelina ISA 315 grafted on Rubidoux The antagonist or synergistic viroids interaction could trifoliate orange inoculated with a viroid mixture carrying also contribute to the understanding of the observed

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Vernière, C., Perrier, X., Dubois, C., Dubois, A., Vidalakis, G., Pagliaccia, D., Bash, J. A., Afunian, M., Botella, L., Chabrier, C., Bové, J. M., & Duran-Vila, & Semancik, J. S. (2011). Citrus dwarfing viroid: effect N. (2006). Interactions between citrus viroids affect on tree size and scion performance specific to Poncirus symptom expression and field performance of Clementine trifoliata rootstock for high density planting. Annals of Applied Biology, (158), 204-217. trees grafted on trifoliate orange. Phytopathology, 96, 356-368. Received: August 28, 2019 Vidalakis, G., Pagliaccia, D., Bash, J. A., & Semancik, Accepted: March 01, 2021 J. S. (2010). Effects of mixtures of citrus viroids as transmissible small nuclear RNA on tree dwarfing and How to cite: Stuchi, E. S., Silva, S. R., Sempionato, O. R., Reiff, E. T., Parolin, commercial scion performance on Carrizo citrange L. G., & Perecin, D. (2021). Viroids and rootstocks on field performance of Tahiti acid lime. Citrus Research & Technology, 41, e1063. https://doi. rootstock. Annals of Applied Biology, 153(3), 415-423. org/10.4322/crt.20420

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